This invention relates to the fields of bioaffinity separations and diagnostic testing of biological samples. More specifically, the invention provides compositions and methods which, may be used in magnetic separation assays and enrichment procedures for controlling endogenous magnetic particle aggregation factors which, if uncontrolled, would obscure visualization of isolated entities. Also provided are methods for constructing and synthesizing reversible aggregation factors and the resulting compositions which simultaneously enhance recovery of rare biological substances while facilitating observation of substances so isolated.
Several publications are referenced in this application by numerals in parentheses in order to more fully describe the state of the art to which this invention pertains. The disclosure of each of these publications is incorporated by reference herein.
Many laboratory and clinical procedures employ bio-specific affinity reactions. Such reactions are commonly utilized in diagnostic testing of biological samples, or for the separation of a wide range of target substances, especially biological entities such as cell, viruses, proteins, nucleic acids and the like. Various methods are available for analyzing or separating the above-mentioned target substances based upon complex formation between the substance of interest and another substance to which the target substance specifically binds. Separation of complexes from unbound material may be accomplished gravitationally, e.g. by settling, or, alternatively, by centrifugation of finely divided particles or beads coupled to the target substance. If desired, such particles or beads may be made magnetic to facilitate the bound/free separation step. Magnetic particles are well known in the art, as is their use in immune and other bio-specific affinity reactions. See, for example, U.S. Pat. No. 4,554,088 and Immunoassavs for Clinical Chemistry, pp. 147-162, Hunter et al. eds., Churchill Livingston, Edinborough (1983). Generally, any material which facilitates magnetic or gravitational separation may be employed for this purpose. However, in the past 20 years the superiority of magnetics for performing such separations has led to its use in many applications.
Magnetic particles generally fall into two broad categories. The first category includes particles that are permanently magnetizable, or ferromagnetic. The second category comprises particles that demonstrate bulk magnetic behavior only when subjected to a magnetic field. The latter are referred to as magnetically responsive particles. Materials displaying magnetically responsive behavior are sometimes described as superparamagnetic. However, materials exhibiting bulk ferromagnetic properties, e.g., magnetic iron oxide, may be characterized as superparamagnetic only when provided in crystals of about 30 nm or less in diameter. Larger crystals of ferromagnetic materials, by contrast, retain permanent magnet characteristics after exposure to a magnetic field and tend to aggregate thereafter due to strong particle-particle interactions. Magnetic particles can be classified as large (1.5 to about 50 microns), small (0.7-1.5 microns), and colloidal or nanoparticles ( less than 200 nm). The latter are also called ferrofluids or ferrofluid-like and have many of the properties of classical ferrofluids. Liberti et al pp 777-790, E. Pelizzetti (ed) xe2x80x9cFine Particles Science and Technologyxe2x80x9d Kluwer Acad. Publishers, Netherlands, 1996.
Small magnetic particles are quite useful in analyses involving bio-specific affinity reactions, as they are conveniently coated with biofunctional polymers (e.g., proteins), provide very high surface areas and give reasonable reaction kinetics. Magnetic particles ranging from 0.7-1.5 microns have been described in the patent literature, including, by way of example, U.S. Pat. Nos. 3,970,518; 4,018,886; 4,230,685; 4,267,234; 4,452,773; 4,554,088; and 4,659,678. Certain of these particles are disclosed to be useful solid supports for immunologic reagents.
In addition to the small magnetic particles mentioned above, there are a class of large magnetic particles ranging in size from approximately 1.5-50 microns, which also have superparamagnetic behavior. Typical of such materials are those invented by Ugelstad (U.S. Pat. No. 4,654,267) and manufactured by Dynal, (Oslo, Norway). The Ugelstad process involves the synthesis of polymer particles which are caused to swell and magnetite crystals are embedded in the swelled particles. Other materials in the same size range are prepared by synthesizing the particle in the presence of dispersed magnetite crystals. This results in the trapping of magnetite crystals in a polymer matrix, thus making the resultant materials magnetic. In both cases, the resultant particles have superparamagnetic behavior, which is manifested by the ability to disperse readily upon removal of the magnetic field. Unlike magnetic colloids or nanoparticles, these materials, as well as small magnetic particles, are readily separated with simple laboratory magnetics because of the mass of magnetic material per particle. Thus, separations are effected in gradients from as low as a few hundred gauss/cm on up to about 1.5 kilogauss/cm. Colloidal magnetic particles, (below approximately 200 nm),on the other hand, require substantially higher magnetic gradients because of their diffusion energy, small magnetic mass per particle and Stokes drag. U.S. Pat. No. 4,795,698 to Owen et al. relates to polymer-coated, colloidal, superparamagnetic particles. Such particles are manufactured by precipitation of a magnetic species in the presence of a biofunctional polymer. The structure of the resulting particles, referred to herein as single-shot particles, has been found to be a micro-agglomerate in which one or more ferromagnetic crystallites having a diameter of 5-10 nm are embedded within a polymer body having a diameter on the order of 50 nm. The resulting particles exhibit an appreciable tendency to remain in aqueous suspension for observation periods as long as several months. U.S. Pat. No. 4,452,773 to Molday describes a material similar in properties to those described in Owen et al., which is produced by forming magnetite and other iron oxides from Fe+2/Fe+3 via base addition in the presence of very high concentrations of dextran. Materials so produced have colloidal properties and have proved to be very useful in cell separation. This technology has been commercialized by Miltenyi Biotec, Bergisch Gladbach, Germany.
Another method for producing superparamagnetic colloidal particles is described in U.S. Pat. No. 5,597,531. In contrast to the particles described in the Owen et al. patent, these latter particles are produced by directly coating a biofunctional polymer onto pre-formed superparamagnetic crystals which have been dispersed, e.g., by sonic energy into quasi-stable crystalline clusters ranging in size from about 25-120 nm. The resulting particles, referred to herein as direct coated (DC) particles, exhibit a significantly larger magnetic moment than Owen et al. or Molday nanoparticles of the same overall size and can be separated effectively in magnetic gradients greater than about 6 kGauss/cm.
Magnetic separation techniques are known wherein a magnetic field is applied to a fluid medium in order to separate ferromagnetic bodies from the fluid medium. In contrast, the tendency of colloidal superparamagnetic particles to remain in suspension, in conjunction with their relatively weak magnetic responsiveness, requires the use of high-gradient magnetic separation (HGMS) techniques in order to separate such particles from a fluid medium in which they are suspended. In HGMS systems, the gradient of the magnetic field, i.e., the spatial derivative, exerts a greater influence upon the behavior of the suspended particles than is exerted by the strength of the field at a given point. High gradient magnetic separation is useful for separating a wide variety of magnetically labeled biological materials, including eukaryotic and prokaryotic cells, viruses, nucleic acids, proteins, and carbohydrates. In methods known heretofore, biological material has been separable by HGMS, provided at least one characteristic determinant is present on the material, which is capable of being specifically recognized and bound to a receptor, such as an antibody, antibody fragment, specific binding protein (e.g., protein A, streptavidin), lectin, and the like. HGMS systems can be divided into two broad categories. One such category includes magnetic separation systems which employ a magnetic circuit that is entirely situated externally to a separation chamber or vessel. Examples of such external separators (or open field gradient separators) are described in U.S. Pat. No. 5,186,827. In several of the embodiments described in the ""827 patent, the requisite magnetic field gradient is produced by positioning permanent magnets around the periphery of a non-magnetic container such that the like poles of the magnets are in a field-opposing configuration. The extent of the magnetic field gradient within the test medium obtainable in such a system is limited by the strength of the magnets and the separation distance between the magnets. Hence, there exists a finite limit to gradients that can be obtained with external gradient systems. In a co-pending application Ser. No. 60/098,021, means for maximizing radial gradients and methods for maximizing separation efficiency via novel vessel designs are disclosed.
Another type of HGMS separator utilizes a ferromagnetic collection structure that is disposed within the test medium in order to: (1) intensify an applied magnetic field; and (2) produce a magnetic field gradient within the test medium. Previously disclosed internal HGMS systems comprise fine steel wool or gauze packed within a column that is situated adjacent to a magnet. The applied magnetic field is concentrated in the vicinity of the steel wires so that suspended magnetic particles will be attracted toward, and adhere to, the surfaces of the wires. The gradient produced on such wires is inversely proportional to the wire diameter whereas the magnetic xe2x80x9creachxe2x80x9d decreases with diameter. Hence, very high gradients can be generated.
One major drawback of internal gradient systems is that the use of steel wool, gauze material, steel microbeads or the like, may entrap non-magnetic components of the test medium by capillary action in the vicinity of intersecting wires or within interstices between intersecting wires. Various coating procedures have been applied to such internal gradient columns (U.S. Pat. Nos. 5,693,539; 4,375,407), however, the large surface area in such systems still creates recovery problems due to absorption. Hence, internal gradient systems are not desirable, particularly when recovery of very low frequency captured entities is the goal of the separation. Further, these systems make automation difficult and costly.
On the other hand, HGMS approaches using external gradients for cell separation provide a number of conveniences. Firstly, simple laboratory tubes such as test tubes, centrifuge tubes or even vacutainers (used for blood collection) can be employed. When external gradients are of the kind in which separated cells are effectively monolayered, as is the case with quadrupole/hexapole devices (U.S. Pat. No. 5,186,827) or the opposing dipole arrangement described in U.S. Pat. No. 5,466,574, washing of cells or subsequent manipulations are facilitated. Further, recoveries of cells from tubes or similar containers is a simple and efficient process. This is particularly the case when compared to recoveries from high gradient columns. Such separation vessels also provide another important feature which is the ability to reduce volume of the original sample. For example, if a particular human blood cell subset, (e.g. magnetically labeled CD 34+ cells), is isolated from blood diluted 20% with buffer to reduce viscosity, a 15 ml conical test tube may be employed as the separation vessel in an appropriate quadrupole magnetic device. After appropriate washes and/or separations and resuspensions to remove non-bound cells, CD34+ cells can very effectively be resuspended in a volume of 200 xcexcl. This can be accomplished, for example, by starting with 12 ml of solution (blood, ferrofluid and dilution buffer) in a 15 ml conical test tube, performing a separation, discarding the xe2x80x9csupernatantxe2x80x9d and subsequent wash xe2x80x9csupernatantsxe2x80x9d and resuspending the recovered cells in 3 ml of appropriate cell buffer. A second separation is then performed which may include additional separation/wash steps (as might be necessary for doing labeling/staining reactions) and finally the isolated cells are easily resuspended in a final volume of 200 xcexcl. By reducing volume in this sequential fashion, and employing a vortex mixer for resuspension, cells adhered to the tube above the resuspension volume are recovered into the reduced volume. When done carefully and rapidly in appropriately treated vessels, cell recovery is quite efficient, ranging between 70-90%.
The efficiency with which magnetic separations can done and the recovery and purity of magnetically labeled cells will depend on many factors. These include such considerations as the number of cells being separated, the receptor density of such cells, the magnetic load per cell, the non-specific binding (NSB) of the magnetic material, the technique employed, the nature of the vessel, the nature of the vessel surface, the viscosity of the medium and the magnetic separation device employed. If the level of non-specific binding of a system is substantially constant, as is usually the case, then as the target population decreases so does the purity. As an example, a system with 0.2% NSB that recovers 80% of a population which is at 0.25% in the original mixture will have a purity of 50%. Whereas if the initial population were at 1.0%, the purity would be 80%. Not as obvious is the fact that the smaller the population of a targeted cell, the more difficult it will be to magnetically label and to recover. Furthermore, labeling and recovery will markedly depend on the nature of magnetic particle employed. For example, when cells are incubated with large magnetic particles, such as Dynal beads, the cells are labeled through collisions created by mixing of the system as the beads tend to be too large to diffuse. Thus, if a cell were present in a population at a frequency of 1 cell/ml of blood or even less, as could be the case for tumor cells in very early cancers, then the probability of labeling target cells will be related to the numbers of magnetic particles added to the system and the length of time of mixing. Since mixing of cells with such particles for substantial periods of time will be deleterious, it becomes necessary to increase particle concentration as much a possible. There is, however, a limit to the quantity of magnetic particle that can be added to the system, in that one can substitute a system comprising a rare cell mixed in with other blood cells with one comprising a rare cell mixed in with large quantities of magnetic particles upon separation, in which case the ability to enumerate the cells of interest or to examine them is not markedly improved.
There is another drawback to the use of large particles to isolate cells having rare frequencies (1-50 cells/ml of blood). Despite the fact that large magnetic particles allow the use of external gradients of very simple design and relatively low magnetic gradient, large particles tend to cluster around cells in a cage-like fashion making them difficult to xe2x80x9cseexe2x80x9d or to analyze. Hence, the particles must be released before analysis, and releasing the particles often introduces other complications.
In theory, colloidal magnetic particles, used in conjunction with high gradient magnetic separation, should be the method of choice for separating a cell subset of interest from a mixed population of eukaryotic cells, particularly if the subset of interest comprises only a small fraction of the entire population. With appropriate magnetic loading, sufficient force is exerted on a cell, facilitating its isolation even in a media as viscous as moderately diluted whole blood. As noted, colloidal magnetic materials below about 200 nanometers will exhibit Brownian motion which markedly enhances their ability to collide with and magnetically label rare cells. This is demonstrated in U.S. Pat. No. 5,541,072, where results of very efficient tumor cell purging experiments are described employing 100 nm colloidal magnetic particles (ferrofluids). Just as importantly, colloidal materials at or below the size range noted do not generally interfere with viewing of cells. Cells so retrieved can be examined by flow cytometry with minimal forward scattering effects or by microscopy employing visible or fluorescent techniques. Because of their diffusive properties, such materials, in contrast to large magnetic particles, readily xe2x80x9cfindxe2x80x9d and magnetically label rare biological entities such as tumor cells in blood.
There is, however, a significant problem which arises in the use of ferrofluid-like materials for cell separation in external field gradient systems which, for reasons given above, is the device design of choice. Direct monoclonal antibody conjugates of Owen et al. materials or Molday nanoparticles, such as those produced by Miltenyi Biotec, do not have sufficient magnetic moment for use in cell selection employing the best available external magnetic gradient devices, such as the quadrupole or hexapole magnetic devices described in U.S. Pat. No. 5,186,827. When used for separations in moderately diluted whole blood, they are even less effective. Using similar materials, which are substantially more magnetic, as described in U.S. Pat. No. 5,698,271, more promising results have been obtained. In model spiking experiments, it has been found that SKBR3 cells (breast tumor line), which have a high EpCAM (epithelial cell-adhesion molecule) determinant density, are efficiently separated from whole blood with direct conjugates of anti EpCAM MAb ferrofluids even at very low spiking densities (1-5 cells/ml blood). On the other hand, PC3 cells (a prostate tumor line) which have low antigen density are separated at significantly lower efficiency. Most likely this is a consequence of inadequate magnetic loading onto these low density receptor cells.
From the foregoing discussion, it would be advantageous to provide a magnetic separation system which combines the beneficial properties of both colloidal magnetic materials and large magnetic particles (e.g., diffusion based labeling and large magnetic moment, respectively) for separations involving rare events or for cells with very low density receptors. One could envision starting a separation process with a magnetic colloidal or nanoparticle which, due to their Brownian motion, would rapidly find and label cells in rare numbers or cells with very low density receptors. Once that labeling is achieved, it would be desirable to convert the magnetic moment of the nanoparticle to a value similar to that of a large magnetic particle. In that way, magnetically labeled entities could be separated in the kinds of gradient fields used for larger particles, e.g., a simple external field gradient separator. In the case of very low density receptor cells, which are recovered inefficiently even in high gradient external field separators, use of such a principle would clearly increase the efficiency of separation. In applications where cells are to be analyzed or used for some biological purpose following separation, it would also be very desirable to be able to convert the magnetic moment of the labeled entity back to that of its original colloidal magnetic labeling density. This approach would permit separation from excessive magnetic material, which would facilitate subsequent analysis or use.
U.S. Pat. No. 5,466,574 to Liberti et al., describes a system which has some of the foregoing features regarding xe2x80x9cloading onxe2x80x9d of magnetic materials onto cells. It was discovered that when cells were first labeled with specific monoclonal antibodies (with or without biotinylation) followed by magnetic labeling with goat anti-mouse ferrofluid or with streptavidin-ferrofluid (respectively), separation was enhanced in the presence of excess monoclonal antibody. The unique ability of ferrofluids to create this xe2x80x9cno washxe2x80x9d enhancing procedure is due to immunochemical crosslinking of free ferrofluid in solution to ferrofluid-bound target cells. Ferrofluid bound to monoclonal antibody on cells, in turn, binds to free ferrofluid in solution via free monoclonal antibody. This results in immunochemical clusters of monoclonal antibody/ferrofluid xe2x80x9cgrowingxe2x80x9d off of monoclonal antibody labeled cell determinants (referred to as chaining). Thus, magnetic colloid is xe2x80x9cartificiallyxe2x80x9d loaded onto cells making them more magnetic and easier to separate. The phenomenon was found to obey immunochemical rules, in that a high excess of monoclonal antibody resulted in a decrease in chaining (monoclonal excess zone) and a loss of separation efficiency. Similarly high levels of ferrofluid also reduced chaining (ferrofluid excess zone). Chaining has been found to be useful for purging unwanted cells, e.g. tumor cells, in bone marrow or peripheral blood xe2x80x9cgrafts.xe2x80x9d By this method, very high levels of magnetic material (visible brown rims around cells, as observed via microscopy) can be loaded onto target cells giving rise to very efficient separation in high gradient fields of only 8-12 kGauss/cm gradients. On the other hand, cells labeled with xe2x80x9cmonomericxe2x80x9d ferrofluid were found to separate less efficiently in the same gradient.
In attempts to use chaining for isolating rare cells from whole blood, several problems have been encountered. First, although spiked cells are, indeed, efficiently recovered, they are so densely covered with ferrofluid (chaining) that the ability to analyze them is markedly reduced. Hence this approach is not ideal for applications wherein the positively selected cells are to be observed via microscopy or flow cytometry. Additionally, chaining seems to promote non-specific binding. In summary, designing a chaining-based assay where the level of chaining simultaneously gives rise to separation enhancement, non-obstructed viewing of the isolated cells and acceptable levels of non-specific binding is extraordinarily difficult. The chaining reaction is difficult to control because it requires immunochemical stoichiometry. For example, most ( greater than 99%) of the added monoclonal antibody (or tagging ligand) will always be free in solution regardless of the affinity of the antibodies. Hence, the amount of ferrofluid required to achieve immunochemical equivalence (where the best separations take place via chaining) generally leads to more chaining than is desired, particularly in the case where the selected cell is to be viewed and/or further studied. Chaining can be lessened by concurrent decreases in labeling monoclonal antibody and added ferrofluid, however this results in a sacrifice of separation efficiency. Another drawback to the use of chaining to enhance separation is the inability to, in some practical manner, reverse chaining. If chaining could be reversed and the concomitant increase in non-specific binding decreased, the phenomenon would provide a viable approach to enabling the desired xe2x80x9cloading onxe2x80x9d of magnetic material. Another disadvantage of this method is that a two step reaction is required, i.e., reaction of targets with primary monoclonal antibody in a first step followed by repetition with ferrofluid specific for primary monoclonal antibody in the second step. This approach cannot be used in assays where primary antibody is directly conjugated to ferrofluid.
U.S. Pat. No. 5,108,933 to Liberti et al. discloses the use of weakly magnetic colloidal materials such as those described by Owen et al. or Molday in immunoassays employing external field magnetic separators. Such materials are described therein as agglomerable and resuspendable colloidal magnetic materials which remain substantially undisturbed in an external magnetic field system, for example, those commercially available at that time (Ciba Corning, Wampole, Mass.; Serono Diagnostics, Norwell, Mass.). By contrast, materials made by the process disclosed in the ""531 patent being substantially more magnetic, as noted above, will separate in those separators. In the ""933 patent means for converting the colloid to an agglomerate are disclosed so as to make them separable in those separators. Thus, such materials could be used for performing the bound/free separation step of immunoassays. There is no mention in ""933 for the need of, or methods for reversing agglomeration reactions.
In light of the foregoing and recent discoveries of naturally occurring ferrofluid aggregation factors, the present inventors have recognized the need for compositions and methods for controlling aggregation of ferrofluid by endogenous factors during the isolation and immunochemical characterization of rare target bioentities. Such compositions and methods may be used to advantage to facilitate analysis and observation of bioentities so isolated. Further, this invention also permits the use of substantially less magnetic reagent as well as the opportunity to use lower magnetic gradients. In the case of a fixed gradient, the invention provides for the capture or isolation of entities which might have otherwise had insufficient magnetic labeling to be captured.
In accordance with the present invention, methods, compositions and kits are provided for controlling the aggregation of ferromagnetic nanoparticles by endogenous aggregation factors. Ferrofluid aggregation often presents problems during subsequent viewing of the isolated targets. The methods of the invention facilitate visualization of the isolated bioentities by allowing the investigator to control the level of aggregation. In one embodiment of the invention, a method is provided for inhibiting the aggregation of magnetic nanoparticles on the surface of isolated target entities. The method comprises obtaining a biological specimen suspected of containing a target bioentity. Next, immunomagnetic suspensions are prepared by mixing the specimen with colloidal, magnetic particles coupled to a biospecific ligand having affinity for at least one characteristic determinant of the target bioentity. The immunomagnetic suspension is thereafter subjected to a magnetic field to obtain target bioentity enriched fractions. Optionally, the fractions are then examined to determine the characteristics of the target bioentity so isolated. Inhibition of ferrofluid aggregation facilitates subsequent analysis of cells as aggregates of ferrofluid on the cell surface are eliminated. The absence of such aggregates is important for several types of analyses including, for example, flow cytometry and immunofluorescence microscopy.
The reagents provided herein efficiently inhibit or remove endogenous aggregation factors. The factor removal or inhibition step may be performed before or simultaneously with the addition of ferrofluid to the biological specimen for separation and enrichment.
To further characterize target bioentities isolated using the methods of the invention, the method optionally includes the steps of adding to the target bioentity enriched fraction at least one biospecific reagent which recognizes and effectively labels at least one additional characteristic determinant on said target bioentity. The labeled target bioentities are then separated in a magnetic field to remove unbound biospecific reagents. A non-cell exclusion agent is added to the separated bioentities to allow exclusion of non-nucleated components present in the sample. After purifying the target bioentity, it is then ready for analysis using a variety of different analysis platforms. Target bioentities include, without limitation, tumor cells, virally infected cells, fetal cells in maternal circulation, virus particles, bacterial cells, white blood cells, myocardial cells, epithelial cells, endothelial cells, proteins, hormones, DNA, and RNA. Target bioentities may be analyzed by a process selected from the group consisting of multiparameter flow cytometry, immunoflourescent microscopy, laser scanning cytometry, bright field base image analysis, capillary volumetry, manual cell analysis and automated cell analysis. Aggregation inhibiting agents suitable for use in the methods of the present invention, include, but are not limited to reducing agents, animal serum proteins, immune-complexes, carbohydrates, chelating agent, unconjugated ferrofluid, and diamino butane. In the case where the endogenous aggregation factor is of the IgM class and reactive with ferrofluids, preferred aggregation inhibiting agents are reducing agents, such as Mercapto ethane sulfonic acid [MES], Mercapto Propane Sulfonic acid [MPS] and dithiothreitol [DTT]. In a particularly preferred embodiment, the biospecific ligand is a monoclonal antibody having affinity for an epithelial cell adhesion molecule.
In an alternative and preferred embodiment of the invention, a method is provided for isolating target bioentities from a biological sample by controlling aggregation of magnetic nanoparticles. The method entails obtaining a biological specimen suspected of containing said target bioentity and contacting the biological specimen with a reagent effective to inactivate any endogenous aggregating factors present. Immunomagnetic suspensions are then prepared wherein the specimen is mixed with colloidal, magnetic particles coupled to a biospecific ligand having affinity for at least one antigen present on the target bioentity, the magnetic particles being further coupled to a first exogenous aggregation enhancing factor which comprises a first member of a specific binding pair. A second multivalent exogenous aggregation enhancing factor is then added to the immunomagnetic suspension to increase aggregation of the particles, the second aggregating enhancing factor comprising the second member of the specific binding pair, which reversibly binds to the magnetically labeled target bioentity. The sample is then subjected to a magnetic field to obtain a target bioentity enriched fraction. This preferred embodiment takes advantage of the fact that aggregating ferrofluid onto target entities in a controlled and reversible fashion results in substantially improved isolation efficiency.
In a further embodiment, the above described method further comprises the steps of adding at least one biospecific reagent which recognizes and labels at least one additional characteristic determinant on said target bioentity. The target bioentity so labeled is then separated in a magnetic field to remove unbound biospecific reagent. A non-cell exclusion agent is added to the separated bioentities to allow exclusion of non-nucleated components present in the sample. The target bioentity is then purified and further analyzed. In order to reverse the aggregation mediated by the exogenous aggregation factors, a member of the specific binding pair may be added in excess to the purified bioentity to reduce ferrofluid aggregation on the surface of cells, thereby facilitating viewing of the cells, e.g. in a microscope. Suitable specific binding pairs for this purpose include, without limitation, biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, Protein A-antibody Fc, avidin-biotin, biotin analog-streptavidin, biotin analog-avidin, desthiobiotin-streptavidin, desthiobiotin-avidin, iminobiotin-streptavidin, and iminobiotin-avidin. Preferably, the biospecific ligand is a monoclonal antibody having affinity for epithelial cell adhesion molecule. Exemplary biospecific reagents include monoclonal antibodies, polyclonal antibodies, detectably labeled antibodies, antibody fragments, and single chain antibodies. Isolated target bioentities may be analyzed by a process selected from the group consisting of multiparameter flow cytometry, immunofluorescent microscopy, laser scanning cytometry, bright field base image analysis, capillary volumetry, manual cell analysis and automated cell analysis.
In accordance with the present invention, controlling aggregation of ferrofluid in a sample has several unexpected benefits previously noted, e.g. increasing efficiency of separation of some particular entity. It has been discovered that addition of an exogenous aggregation enhancing factor gives rise to increased magnetic loading, resulting in increased separation efficiency while reducing the amount of ferrofluid required to isolate the target bioentity. The increased magnetic loading also allows for reduced incubation periods and facilitates isolation of the target bioentity in the presence of a suboptimal magnetic field.
In an additional embodiment of the present invention a kit is provided which facilitates the practice of the methods described herein. An exemplary kit for isolating target bioentities includes i) coated magnetic nanoparticles comprising a magnetic core material, a protein base coating material, and an antibody that binds specifically to a first characteristic determinant of said target bioentity, said antibody being coupled, directly or indirectly, to said base coating material; ii) at least one antibody having binding specificity for a second characteristic determinant of said rare biological substance; iii) an aggregation inhibiting factor; and iv) a non-cell exclusion agent for excluding non-nucleated sample components other than said target bioentity from analysis.
A kit for improving the isolation efficiency of certain biological entities, such as might be required for isolating low antigen density tumor cells from a biological sample, is also provided in accordance with the present invention. This kit utilizes controlled and reversible aggregation of magnetic nanoparticles to achieve such improvement. Such a kit includes i) a reagent effective to inactivate endogenous aggregating factors; ii) coated magnetic nanoparticles comprising a magnetic core material, a protein base coating material, and an antibody that binds specifically to a first characteristic determinant of said tumor cell, the antibody being coupled, directly or indirectly, to the base coating material; the magnetic particles being further coupled to a first exogenous aggregation enhancing factor, the factor comprising one member of a specific binding pair; iii) at least one antibody having binding specificity for a second characteristic determinant of said tumor cell; iv) a second exogenous aggregation enhancing factor, the second aggregation enhancing factor comprising the second member of the specific binding pair; and v) a non-cell exclusion agent for excluding non-nucleated sample components other than the tumor cells from analysis. The kit may optionally include a reagent for reversing the exogenous aggregation factor. Specific binding pairs useful in such a kit, include without limitation, biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, Protein A-antibody Fc, and avidin-biotin, biotin analog-avidin, desthiobiotin-streptavidin, desthiobiotin-avidin, iminobiotin-streptavidin, and iminobiotin-avidin. Reagents effective to inactivate endogenous aggregating factors include reducing agents, animal serum proteins, immune-complexes, carbohydrates, chelating agent, unconjugated ferrofluid, and diamino butane.
The methods, compositions and kits of the invention provide the means for controlling the aggregation of magnetic nanoparticles, thus facilitating the isolation, visualization and characterization of rare biological substances or cells from biological specimens.